It has been reported that 4-1BB triggering in vivo selectively suppressed the recall response of staphylococcal enterotoxin A (SEA)-specific CD4(+) T cells, in which CD8(+) T-derived TGF-beta was involved. Here, we have examined an alternative mechanism for the 4-1BB-mediated CD4(+) T suppression, as the neutralization of TGF-beta is only effective in rescuing the SEA-specific recall response at high cellular concentrations. We show that this selective suppression of CD4(+) T cells by 4-1BB triggering in vivo is mediated mainly by induction of indoleamine 2,3-dioxygenase (IDO) in an IFN-gamma-dependent manner. SEA-specific CD4(+) T responses were suppressed partly by TGF-beta-expressing CD8(+) T cells, particularly CD11c(+)CD8(+) T cells, but strongly inhibited by dendritic cells (DCs) expressing IDO. IFN-gamma that increased IDO in DCs was produced primarily from CD11c(+)CD8(+) T cells, which were expanded selectively by 4-1BB stimulation. CD4(+), CD8(+), and plasmacytoid DCs exerted a similar suppressive activity toward the SEA-specific CD4(+) T cells. Neutralization of IFN-gamma or IDO activity in vivo largely reversed the 4-1BB-mediated CD4(+) T suppression. Collectively, these data indicate that 4-1BB-dependent suppression of SEA-specific CD4(+) T responses was mediated mainly by IFN-gamma-dependent IDO induction and partially by TGF-beta.